Site-specific conjugation of linker drugs to antibodies and resulting ADCS

ABSTRACT

The present invention relates to antibody-drug conjugates (ADCs) wherein a linker drug is site-specifically conjugated to an antibody through an engineered cysteine, and their use as a medicament, notably for the treatment of human solid tumours and haematological malignancies, in particular breast cancer, gastric cancer, colorectal cancer, urothelial cancer, ovarian cancer, uterine cancer, lung cancer, mesothelioma, liver cancer, pancreatic cancer, prostate cancer, and leukaemia.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional under 35 U.S.C. § 120 ofcopending U.S. application Ser. No. 15/312,436, filed Nov. 18, 2016,which is a U.S. national stage application under 35 U.S.C. § 371 of PCTInternational Application No. PCT/EP2015/061456 filed May 22, 2015,which claims the benefit of priority to European Application No.14169493.5, filed on May 22, 2014; the disclosure of each prior U.S.application is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“P1664US01_PB-067_ST25.txt” created on Aug. 19, 2019 and is 27,046 bytesin size. The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to antibody-drug conjugates (ADCs) whereina linker drug is site-specifically conjugated to an antibody through anengineered cysteine, and their use in the treatment of human solidtumours and haematological malignancies, in particular breast cancer,gastric cancer, colorectal cancer, urothelial cancer, ovarian cancer,uterine cancer, lung cancer, mesothelioma, liver cancer, pancreaticcancer, prostate cancer, and leukaemia.

BACKGROUND OF THE PRESENT INVENTION

Antibody-drug conjugates (ADCs) are an emerging class of targetedtherapeutics having an improved therapeutic index over traditionalchemotherapy. Drugs and linkers have been the focus of ADC development,in addition to (monoclonal) antibody (mAb) and target selection.Recently, however, the importance of conjugate homogeneity was realized.The conventional methods for drug attachment to an antibody lead to aheterogeneous mixture, and some individual constituents of that mixturecan have poor in vivo performance. Newer methods for site-specific drugattachment lead to more homogeneous conjugates and allow control of thesite of drug attachment. These subtle improvements can have profoundeffects on in vivo efficacy and/or in vivo safety and thereby on thetherapeutic index. Methods for site-specific drug conjugation toantibodies are comprehensively reviewed by C. R. Behrens and B. Liu inmAbs, Vol. 6, Issue 1, 2014, pages 1-8.

Conventional ADCs are typically produced by conjugating the linker drugto the antibody through the side chains of either surface-exposedlysines or free cysteines generated through reduction of interchaindisulfide bonds. Because antibodies contain many lysine residues andcysteine disulfide bonds, conventional conjugation typically producesheterogeneous mixtures that present challenges with respect toanalytical characterization and manufacturing. Furthermore, theindividual constituents of these mixtures exhibit differentphysicochemical properties and pharmacology with respect to theirpharmacokinetic, efficacy, and safety profiles, hindering a rationalapproach to optimizing this modality.

These two conventional techniques for chemical modification ofantibodies were used to construct the two ADCs with current FDAmarketing approvals. Brentuximab vedotin (Adcetris™, Seattle Genetics)consists of an anti-CD30 monoclonal antibody conjugated to the highlycytotoxic drug monomethyl auristatin E (MMAE) via modification of nativecysteine side chain thiols. The manufacture involves partial reductionof the solvent-exposed interchain disulfides followed by modification ofthe resulting thiols with maleimide-containing linker drugs. Forbrentuximab vedotin, the thiols were modified with mc-vc-PAB-MMAE, whichincorporates a cathepsin B protease cleavage site (vc,valine-citrulline) and a self-immolative linker (PAB,para-aminobenzyloxycarbonyl) between the maleimide group (mc,maleimidocaproyl) and the cytotoxic drug (MMAE). The cysteine attachmentstrategy results in maximally two drugs per reduced disulfide. Mosthuman IgG molecules have four solvent-exposed disulfide bonds, and so arange of from zero to eight drugs per antibody is possible. The exactnumber of drugs per antibody is determined by the extent of disulfidereduction and the number of molar equivalents of linker drug used in theensuing conjugation reaction. Full reduction of all four disulfide bondsgives a homogeneous construct with eight drugs per antibody, while apartial reduction typically results in a heterogeneous mixture withzero, two, four, six, or eight drugs per antibody. Brentuximab vedotinhas an average of about 4 drugs per antibody.

The other ADC with current FDA approval is ado-trastuzumab emtansine(T-DM1, Kadcyla™, Roche/Genentech), which was constructed by couplingthe anti-HER2 monoclonal antibody trastuzumab to the cytotoxic drugmaytansine through modification of lysine side chain amines. Thisversion of maytansine (DM1) was modified to include a thiol that couldbe attached to a maleimide linker. A bifunctional linker (SMCC,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) with amaleimide at one end and an N-hydroxysuccinimidyl (NHS) ester at theother end was reacted with lysine primary amine side chains to form astable amide bond. The modified maytansine (DM1) was then attached tothe antibody through conjugation to the maleimide end of thebifunctional linker. In contrast to the linker utilized in brentuximabvedotin, this linker has no (protease) cleavage site and thus requireslysosomal degradation of the antibody part of the ADC to liberate theactive DM1-linker-lysine metabolite. The attachment method resulted in aheterogeneous mixture of conjugates with an average of 3.5 drugs perantibody. Compared with the cysteine method described above, thisstrategy gave a more heterogeneous mixture because 20 to 40 lysineresidues were found to be modified, whereas only maximally 8 differentcysteine residues are modified using the native cysteine modificationmethod.

Recently, it was reported that the pharmacological profile of ADCs maybe improved by applying site-specific conjugation technologies that makeuse of surface-exposed cysteine residues engineered into antibodies thatare then conjugated to a linker drug, resulting in site-specificallyconjugated ADCs with defined drug-to-antibody ratios (DARs). Relative tothe heterogeneous mixtures created using conventional lysine andcysteine conjugation methodologies, site-specifically conjugated ADCshave generally demonstrated at least equivalent in vivo potency,improved PK, and an expanded therapeutic window.

The first site-specific conjugation approach was developed at Genentechby introducing a cysteine residue using site-directed mutagenesis atpositions showing high thiol reactivity as elaborated in WO2006/034488.This common practice in protein modification was more complicated in anantibody because of the various native cysteine residues alreadypresent. Introducing the extra cysteine residue in an unsuitableposition could result in improper formation of interchain disulfidebonds and therefore improper folding of the antibody. Engineeredcysteine residues in suitable positions in the mutated antibody areoften capped by other thiols, such as cysteine or glutathione, to formdisulfides.

Drug attachment to the mutant residues was achieved by reducing both thenative interchain and mutant disulfides, then re-oxidizing the nativeinterchain cysteines using a mild oxidant such as CuSO₄ ordehydroascorbic acid, followed by standard conjugation of the liberatedmutant cysteine with a linker drug. Under optimal conditions, two drugsper antibody will be attached (if one cysteine is engineered into theheavy chain or light chain of the mAb). The engineered cysteine methodproved to be suitable for developing the site-specific ADC SGN-CD33A(Seattle Genetics), which recently entered a Phase I dose-escalationclinical study as a treatment for acute myeloid leukaemia (AML), as wellas a Phase Ib clinical trial in combination with standard of carechemotherapy, including cytarabine and daunorubicin. This ADC comprisesa cleavable dipeptide linker (i.e., valine-alanine) and aDNA-cross-linking, pyrrolobenzodiazepine (PBD) dimer as the drug linkedto heavy chain position S239C in the Fc part of IgG1 mAb h2H12 (DAR 1.9;Sutherland et al. Blood 2013; 122(8):1455-1463).

Whereas in WO2006/034488 specifically surface accessible valine, alanineand serine residues not involved in antigen binding interactions anddistant from the existing interchain disulfide bonds were substituted toobtain engineered cysteine residues with high thiol reactivity,WO2014/124316 from Novartis specifically focuses on the identificationof surface accessible sites in the constant regions of the antibodyheavy and light chains, at which sites substitution for a cysteineresidue enables efficient conjugation of payloads and providesconjugates with high stability.

In addition to the engineered cysteine conjugation strategy, othermethods for site-specific attachment of drugs have been developed.Pfizer demonstrated a new technique for conjugation using microbialtransglutaminase to couple an amine-containing drug to an engineeredglutamine on the antibody. Transglutaminase is an enzyme that catalyzesamide bond formation between the acyl group of a glutamine side chainand the primary amine of a lysine side chain.

In addition to enzymatic conjugation, orthogonal chemistry conjugationhas also been used to site-specifically modify a wide variety ofproteins using non-natural amino acids (notably technologies from Ambrxand Sutro Biopharma). In particular, p-acetylphenyl-alanine andp-azidomethyl-L-phenylalanine were chosen as the non-natural aminoacids, because they, respectively, contain a ketone and an azidefunctional group that is not found in any of the 20 natural amino acidside chains. This allows for specific modification of the ketone cq.azide groups without interference from other amino acids. This methodprovided an additional route for constructing ADCs with a maximum of twodrugs per antibody (per one such non-natural amino acid).

In all of the prior art methods disclosed thus far, the emphasis was puton site-specifically conjugating linker drugs at surface/solvent-exposedpositions, at positions showing high thiol reactivity, and at positionsin specifically the constant regions of monoclonal antibodies, with theaim of improving homogeneity and pharmacokinetic properties. Even thoughthe above-described conventional lysine and cysteine conjugation methodshave led to FDA-approved antibody-drug conjugates and they are beingused for constructing most of a large number of ADCs currently inpreclinical and clinical trials, there is still a need for newconjugation strategies with the aim to (further) improve thephysicochemical, pharmacokinetic, pharmacological, and/or toxicologicalproperties of ADCs to obtain ADCs having acceptable antigen bindingproperties, in vivo efficacy, therapeutic index, and/or stability.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to antibody-drug conjugates (ADCs) whereina linker drug is site-specifically conjugated to an antibody through anengineered cysteine at one or more specific positions of said antibody,and their use in the treatment of human solid tumours and haematologicalmalignancies, in particular breast cancer, gastric cancer, colorectalcancer, urothelial cancer, ovarian cancer, uterine cancer, lung cancer,mesothelioma, liver cancer, pancreatic cancer, prostate cancer, andleukaemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Identification of suitable linker drug conjugation positions inthe Fab part of an antibody

FIG. 1B. Docking of duocarmycin linker drug vc-seco-DUBA in the Fabcavity of an antibody (overlay of multiple vc-seco-DUBA dockings)

FIG. 1C. Identification of suitable linker drug conjugation positions inthe Fc part of an antibody

FIG. 1D. Docking of duocarmycin linker drug vc-seco-DUBA in the Fccavity of an antibody (overlay of multiple vc-seco-DUBA dockings) FIG.2A. In vivo efficacy of engineered cysteine anti-PSMA (VH S41C) ADC(SYD1091) versus vehicle control, comparator engineered cysteineanti-PSMA (CH T120C) ADC (SYD1035), and non-engineered anti-PSMA(wild-type) wt ADC (SYD998) at 2 mg/kg each

FIG. 2B. In vivo efficacy of engineered cysteine anti-PSMA (VH S41C) ADC(SYD1091) versus vehicle control, comparator engineered cysteineanti-PSMA (CH T120C) ADC (SYD1035), and non-engineered anti-PSMA wt ADC(SYD998) at 10 mg/kg each

FIG. 3. Effect on body weight of engineered cysteine anti-PSMA (VH S41C)ADC (SYD1091) versus vehicle control, comparator engineered cysteineanti-PSMA (CH T120C) ADC (SYD1035), and non-engineered anti-PSMA wt ADC(SYD998) at 10 mg/kg each

FIG. 4A. In vivo efficacy of engineered cysteine anti-5T4 (VH P41C) H8ADC (H8-41C-vc-seco-DUBA) versus vehicle control, and non-engineeredanti-5T4 wt H8 ADC (H8-vc-seco-DUBA) at 3 mg/kg each

FIG. 4B. In vivo efficacy of engineered cysteine anti-5T4 (VH P41C) H8ADC (H8-41C-vc-seco-DUBA) versus vehicle control, and non-engineeredanti-5T4 wt H8 ADC (H8-vc-seco-DUBA) at 10 mg/kg each

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Antibody-drug conjugates (ADCs) are emerging as a new class ofanticancer therapeutics that combine the efficacy of small-moleculetherapeutics with the targeting ability of antibodies. By combiningthese two components into a single new molecular entity, highlycytotoxic small molecule drugs can be delivered to cancerous targettissues, thereby enhancing efficacy while reducing the potentialsystemic toxic side effects of the small molecule.

Antibodies have been conjugated to a variety of cytotoxic drugs,including small molecules that bind DNA (e.g. anthracyclines), alkylateor crosslink DNA (e.g. duocarmycins or pyrrolobenzodiazepine dimers,respectively), cause DNA strand breaks (e.g. calicheamicins) or disruptmicrotubules (e.g. maytansinoids and auristatins).

The present invention relates to an antibody-drug conjugate (ADC)compound wherein a linker drug is site-specifically conjugated to anantibody through an engineered cysteine at one or more positions of saidantibody selected from heavy chain 40, 41, 89 (Kabat numbering), 152,153, 155, 171, 247, 297, 339, 375 and 376 (Eu numbering), and lightchain 40, 41 (Kabat numbering), 165 and 168 (Eu numbering).

In one embodiment, the present invention relates to an antibody-drugconjugate (ADC) compound wherein a linker drug is site-specificallyconjugated to an antibody through an engineered cysteine at one or morepositions of said antibody selected from heavy chain 40, 41, 89, 152,153, 155, 171, 247, 297, 339 and 375, and light chain 40, 41, and 165.

In a particularly preferred embodiment, the present invention relates toan antibody-drug conjugate compound wherein a linker drug issite-specifically conjugated to an antibody through an engineeredcysteine at one or more positions of said antibody selected from heavychain 40, 41 and 89 (according to Kabat numbering) and light chain 40and 41 (according to Kabat numbering).

As the focus in earlier work on site-specific ADCs was on findingconjugation positions that show good reactivity with the linker drug,and at the same time have a low risk of forming disulfide bonds betweenantibodies (leading to aggregation) or disturbing the antibody structure(so-called disulfide bridge shuffling), the effects on hydrophobicity ofthe conjugates in relation to the conjugation site have not beenevaluated. In addition, the focus has primarily been on finding suitablesites in the constant regions of the antibody, as modification of thevariable regions of an antibody is generally thought to be associatedwith a high risk of partial or complete loss of antigen binding.

The current inventors, however, have focused on influencing thehydrophobicity characteristics of site-specific ADCs.

An in silico method, employing the YASARA software package (yasara.org,see: Krieger et al. Proteins 2009; 77 Suppl 9: 114-122), was used toidentify sites of strong interaction of the linker drug with theantibody. Suitable locations show a minimal increase in the hydrophobicsurface. In the vicinity of the thus-identified interaction sitessuitable residues (i.e., with sufficient accessibility) to convert tocysteines were identified. In this approach no limitation was made tothe constant regions of the antibody, also the variable region aminoacids were considered if not in the vicinity of antigen binding sites.Locations in the variable domain of the Fab part turned out to bepreferable.

Docking of linker drugs into the Fab and Fc models of various antibodieswas simulated with the commonly used VINA algorithm (Trott O and Olson AJ. J. Comput. Chem. 2010; 31: 455-461) as implemented in YASARA. Theantibody Fab and Fc models used were obtained from X-ray structures orby homology modeling using YASARA.

The duocarmycin type linker drugs, e.g. vc-seco-DUBA (i.e., SYD980; anADC compound thereof is depicted in formula II), were shown to have astrong preference for binding in cavities which are present in allantibody structures (see FIGS. 1B and 1D for the Fab and the Fc part ofan antibody, respectively). Multiple suitable conjugation positions forlinker drug attachment were identified in and in close proximity tothese cavities, i.e., with good accessibility of engineered cysteines atthese locations (see FIGS. 1A and 1C for the Fab and the Fc part of anantibody, respectively).

In the context of the present invention, Kabat numbering is used forindicating the amino acid positions of engineered cysteines in the heavychain (HC) and light chain (LC) variable regions and Eu numbering isused for indicating the positions in the heavy chain and light chainconstant regions of the antibody. In view of the sequence variability inthe variable regions of antibodies, the exact amino acid to besubstituted by cysteine can be different for different antibodies. Formost antibodies, in particular IgG antibodies, in the heavy chain of thevariable region (VH), there usually is an A or S at position 40, a P atposition 41 and a V at position 89 and in the light chain of thevariable region (VL), there usually is a P at position 40 and a G atposition 41. In the heavy chain of the constant regions (CH1, CH2 andCH3), there is normally an E at position 152, a P at position 153, a Tat position 155, a P at position 171, a P at position 247, an N atposition 297, an A at position 339, an S at position 375 and a D atposition 376, and in the light chain of the κ constant region (CL),there is normally an E at position 165 and an S at position 168. In thefive λ light chain isotype constant regions (CL), there is normally an Sat position 165 and an S at position 168.

The expression “Kabat numbering” refers to the numbering system used forheavy chain variable domains or light chain variable domains of thecompilation of antibodies in Kabat, E. A. et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). Using this numbering system,the actual linear amino acid sequence may contain fewer or additionalamino acids corresponding to a shortening of, or insertion into, aframework region (FR) or complementary determining region (CDR) of thevariable domain. The Kabat numbering of residues may be determined for agiven antibody by alignment at regions of homology of the sequence ofthe antibody with a “standard” Kabat numbered sequence.

The expression “Eu numbering” refers to the Eu index as in Kabat, E. A.et al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., NIHpublication no. 91-3242, pp. 662, 680, 689 (1991). The “Eu index as inKabat” refers to the residue numbering of the human IgG1 Eu antibody(Edelman, G. M. et al., Proc. Natl. Acad. Sci. USA, 63, 78-85 (1969)).

Heavy chain positions 40, 41 and 89 are located in the variable regionand positions 152, 153, 155, 171, 247, 297, 339, 375 and 376 are locatedin the constant region of the antibody. Light chain positions 40 and 41are located in the variable region and positions 165 and 168 are locatedin the constant region of the antibody.

Heavy chain positions 40, 41, 89, 152, 153, 155 and 171 and light chainpositions 40, 41, 165 and 168 are located in the Fab part and heavychain positions 247, 297, 339, 375 and 376 are located in the Fc part ofthe antibody.

In accordance with the present invention, the term “engineered cysteine”means replacing a non-cysteine amino acid in the heavy chain or lightchain of an antibody by a cysteine. As is known by the person skilled inthe art, this can be done either at the amino acid level or at the DNAlevel, e.g. by using site-directed mutagenesis.

The present inventors surprisingly have found that the site-specificallyconjugated ADC compounds of the present invention show improvedphysicochemical, pharmacological and/or pharmacokinetic properties, ascompared to ADCs wherein the linker drug is conjugated through nativeinterchain disulfide bonds of the antibody and, moreover, as compared toengineered cysteine ADCs wherein the linker drug is conjugated atpositions disclosed in the prior art from the ones specifically claimedin this patent application. The ADC compounds in accordance with thepresent invention have binding properties similar to the nakedantibodies, good in vivo efficacy, an increased therapeutic index and/orimproved stability. Notably, it was found that the ADC compounds aregenerally less hydrophobic and less susceptible to cathepsin B cleavageand therefore likely also to other intra- or extracellularenzymes/proteases in the tumour mass (tumour microenvironment) than ADCsthat are site-specifically conjugated at different positions, but stillshow similar in vitro cytotoxicity. Unexpectedly, ADCs in accordancewith the present invention show improved in vivo efficacy in a tumourxenograft animal model as compared to ADCs that are site-specificallyconjugated at other positions.

Without wishing to be bound by any theory, the present inventors havefound that when linker drugs are conjugated at the specific positions ofthe antibody as claimed herein, said linker drug fits into either theFab cavity that is formed by the CH1, VH, VL and CL domains of theantibody or the Fc cavity that is formed by the two CH2 and two CH3domains of the antibody. In an IgG1 antibody the top of the Fc cavity isformed by the glycoside/carbohydrate that is attached to the heavy chainposition N297. As a result, the linker drug (which typically is morehydrophobic than the antibody) is shielded from the hydrophilic aqueousenvironment surrounding the antibody and the ADC as such is lesshydrophobic as compared to ADCs wherein the linker drug is conjugatedthrough native disulfide bonds of the antibody and is much lesshydrophobic as compared to ADCs wherein the linker drug issite-specifically conjugated at different positions that are notpresently claimed and where the linker drug is forced to the outside ofthe antibody, i.e., is pointed in a direction away from the antibody.

In one particular embodiment, the present invention relates to anantibody-drug conjugate (ADC) compound wherein a linker drug issite-specifically conjugated to an antibody through an engineeredcysteine at one or more positions of said antibody selected from heavychain 40, 41, 152, 153, 247, 339 and 375, and light chain 40, 41, and165.

In another embodiment, the present invention relates to an antibody-drugconjugate (ADC) compound wherein a linker drug is site-specificallyconjugated to an antibody through an engineered cysteine at one or morepositions of said antibody selected from heavy chain 40, 41, 89, 247,297 and 376, and light chain 40 and 41.

In one embodiment, the present invention relates to an antibody-drugconjugate (ADC) compound wherein a linker drug is site-specificallyconjugated to an antibody through an engineered cysteine at one or morepositions of said antibody selected from heavy chain 40, 41, 89, 152,153, 155 and 171, and light chain 40, 41, 165 and 168 in the Fab part ofsaid antibody.

In another embodiment, the present invention relates to an antibody-drugconjugate (ADC) compound wherein a linker drug is site-specificallyconjugated to an antibody through an engineered cysteine at one or morepositions of said antibody selected from heavy chain 40, 41, 152 and153, and light chain 40, 41 and 165 in the Fab part of said antibody.

Modification of the variable part of an antibody is generally avoided asit can lead to partial or complete loss of antigen binding properties.However, contrary to the general expectations, it was found thatspecific residues in the framework regions of the heavy and light chainsof the antibody are both suitable for conjugation and do not lead to(significant) reduction of antigen binding after conjugation of thelinker drug. Therefore, in a particularly preferred embodiment, thepresent invention relates to an antibody-drug conjugate (ADC) compoundwherein said engineered cysteine is at one or more positions of saidantibody selected from heavy chain 40, 41 and 89 and light chain 40 and41 in the Fab part of said antibody. Preferably, said engineeredcysteine is at heavy chain position 40 or 41 and/or light chain position40 or 41, more preferably at heavy chain position 41 and/or light chainposition 40 or 41, most preferably at heavy chain position 41. As it isknown from the literature that tumour-associated proteases in the tumourmicroenvironment can partially cleave the Fc constant domains, under thehinge region, conjugation in the Fab part is preferred over conjugationin the Fc part. Cleavage of the Fc constant domains would result in lossof Fc-conjugated linker drugs, which in turn could lead to a decreasedactivity of the ADC in vivo. (Fan et al. Breast Cancer Res. 2012; 14:R116 and Brersky et al. PNAS 2009; 106: 17864-17869). Moreover,conjugation to these positions in the Fab part also enables the use ofantigen binding fragments.

In a specific embodiment, the antibody-drug conjugate (ADC) compound ofthe above preferred embodiment may further comprise an additionalengineered cysteine at one or more positions of the antibody selectedfrom heavy chain 152, 153, 155, 171, 339 and 375, and light chain 165and 168. Preferably said further engineered cysteine is at heavy chainposition 375 in the Fc part of said antibody.

In accordance with the present invention, the one or more cysteineresidues can be engineered into the antibody by using conventionalmolecular cloning techniques or the heavy chain or light chain domain(s)of the antibody carrying the cysteine mutation(s) can be synthesized assuch using known (peptide or DNA) synthesis equipment and procedures.

In accordance with the present invention, any linker drug known in theart of ADCs can be used for site-specific conjugation to an antibody,provided it has a chemical group which can react with the thiol group ofan engineered cysteine, typically a maleimide or haloacetyl group.Suitable linker drugs may comprise a duocarmycin, calicheamicin,pyrrolobenzodiazepine (PBD) dimer, maytansinoid or auristatin derivativeas a cytotoxic drug. Either a cleavable or a non-cleavable linker may beused in accordance with the present invention. Suitable examples ofmaytansinoid drugs include DM1 and DM4. Suitable examples of auristatindrugs include MMAE and MMAF.

These abbreviations are well-known to the skilled artisan. Examples ofsuitable linker drugs known to the person skilled in the art includemc-vc-PAB-MMAE (also abbreviated as mc-vc-MMAE and vc-MMAE), mc-MMAF,and mc-vc-MMAF. Preferably, the linker used is a cleavable linker, suchas valine-citrulline (vc) or valine-alanine (va).

The generic molecular structures of a vc-MMAE ADC and mc-MMAF ADC aredepicted below.

In one embodiment, the present invention relates to an ADC compoundwherein said linker drug comprises a duocarmycin derivative.

Duocarmycins, first isolated from a culture broth of Streptomycesspecies, are members of a family of antitumour antibiotics that includeduocarmycin A, duocarmycin SA, and CC-1065. These extremely potentagents allegedly derive their biological activity from an ability tosequence-selectively alkylate DNA at the N3 position of adenine in theminor groove, which initiates a cascade of events that terminates in anapoptotic cell death mechanism.

WO2011/133039 discloses a series of linker drugs comprising aduocarmycin derivative of CC-1065. Suitable linker-duocarmycinderivatives to be used in accordance with the present invention aredisclosed on pages 182-197. The chemical synthesis of a number of theselinker drugs is described in Examples 1-12 of WO2011/133039.

In one embodiment, the present invention relates to a compound offormula (I)

wherein

n is 0-3, preferably 0-1,

m represents an average DAR of from 1 to 6, preferably of from 1 to 4,

R¹ is selected from

y is 1-16, and

R² is selected from

In the structural formulae shown in the present specification, nrepresents an integer from 0 to 3, while m represents an averagedrug-to-antibody ratio (DAR) of from 1 to 6. As is well-known in theart, the DAR and drug load distribution can be determined, for example,by using hydrophobic interaction chromatography (HIC) or reversed phasehigh-performance liquid chromatography (RP-HPLC). HIC is particularlysuitable for determining the average DAR.

Compounds of the formula (I) in accordance with the present inventioncan be obtained according to methods and procedures that are well knownto a person skilled in the art. Suitable methods for site-specificallyconjugating linker drugs can for example be found in Examples 7 and 8 ofWO2005/084390, which describe complete reduction strategies for(partial) loading of antibodies with the linker drug vc-MMAE, inExamples 11 and 12 of WO2006/034488, which describe site-specificconjugation of a maytansine (DM1)-comprising linker drug, and inDoronina et al. Bioconjugate Chem. 17 (2006): 114-124, which describesthe conjugation with mc-MMAF.

Conjugation to two or more of the engineered cysteine sites of thepresent invention allows for the preparation of ADCs comprisinghydrophobic drug classes with a higher DAR, notably DAR 4, withoutgetting too much aggregate.

In accordance with a particular embodiment of the present invention, oneor two engineered cysteines can be incorporated into the heavy chainand/or light chain of the antibody, under optimal reaction conditionsresulting in an ADC compound having a DAR of 2 or 4, respectively. Whenone engineered cysteine is introduced, it can be located either in theFab or in the Fc part of the antibody. It is preferred to introduce saidcysteine in the Fab part of the antibody at position HC 40, 41, 89, 152or 153 or LC 40, 41 or 165, preferably HC 40, 41 or 89 or LC 40, 41 or165, more preferably HC 40 or 41 or LC 40 or 41, even more preferably HC41 or LC 40 or 41, most preferably HC 41. When two engineered cysteinesare introduced, these two cysteines can both be located in the Fab or inthe Fc part of the antibody or, preferably, one can be in the Fab part,preferably HC 40, 41, 152 or 153 or LC 40, 41 or 165, more preferably HC40 or 41 or LC 40 or 41, even more preferably HC 41 or LC 40 or 41, mostpreferably HC 41, and the other can be in the Fc part of the antibody,preferably HC 247, 297, 339 or 375, more preferably HC 339 or 375, mostpreferably HC 375. When two engineered cysteines are introduced in theFab part of the antibody, one cysteine residue may be introduced in theheavy chain and the other cysteine is introduced in the light chain ofthe antibody, e.g. HC 40 or 41 and LC 40 or 41. In addition, when twoengineered cysteines are introduced in the Fab part of the antibody, onecysteine residue may be introduced at one of the specific positions asidentified in the present invention, e.g. HC 40 or 41 or LC 40 or 41,and the other may be located at a surface-exposed (i.e., not hereinclaimed) engineered cysteine position leading to a higher DAR and stillacceptable hydrophobicity.

In a particular embodiment, the present invention relates to a compoundof the formula (I) as disclosed hereinabove, wherein n is 0-1, mrepresents an average DAR of from 1 to 6, preferably of from 1 to 4,more preferably of from 1 to 2, most preferably of from 1.5 to 2,

R¹ is selected from

y is 1-16, preferably 1-4, and

R² is selected from

In a specific embodiment, the present invention relates to a compound ofthe structural formula (I) as disclosed hereinabove, wherein n is 0-1, mrepresents an average DAR of from 1.5 to 2, R¹ is

y is 1-4, and R² is selected from

In a particularly preferred embodiment, the present invention relates toa compound of formula (II)

In accordance with the present invention, any antibody—particularly anyantibody known to have therapeutic activity or any antibody known in theart of ADCs—or any antigen binding fragment thereof can be used forsite-specific conjugation of a linker drug at the specific antibodypositions claimed herein. Said antibody can be an IgA, IgD, IgE, IgG orIgM antibody. Said antibody can have κ (kappa) light chains or λ(lambda) light chains. Said IgG antibody can be an IgG1, IgG2, IgG3 orIgG4 antibody. Preferably, the antibody binds to a(n) antigen targetthat is expressed in or on the cell membrane (e.g., on the cell surface)of a tumour cell, more preferably, the antibody is internalised by thecell after binding to the (antigen) target, after which the toxin isreleased intracellularly. Preferably, the antibody is an IgG antibody,more preferably an IgG1 antibody, most preferably an IgG1 antibodyhaving κ light chains. Preferably, the IgG antibody carries a nativeglycoside/carbohydrate moiety attached at N297 of the heavy chain of theantibody.

Suitable antibodies include an anti-annexin A1 antibody, an anti-CD19antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33antibody, an anti-CD37 antibody, an anti-CD38 antibody, an anti-CD44antibody, an anti-CD47 antibody, an anti-CD56 antibody, an anti-CD70antibody, an anti-CD74 antibody, an anti-CD79 antibody, an anti-CD115antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti-CD203cantibody, an anti-CD303 antibody, an anti-CEACAM antibody, an anti-CLL-1antibody, an anti-c-MET (or anti-HGFR) antibody, an anti-Criptoantibody, an anti-DLL3 antibody, an anti-EGFR antibody, an anti-EPCAMantibody, an anti-EphA2 antibody, an anti-EphB3 antibody, an anti-ETBRantibody, an anti-FcRLS antibody, an anti-FOLR1 antibody, an anti-GCCantibody, an anti-GPNMB antibody, an anti-Her2 antibody, an anti-HMW-MAAantibody, an anti-integrin antibody, an anti-Lewis A like carbohydrateantibody, an anti-Lewis Y antibody, an anti-LIV1 antibody, ananti-mesothelin antibody, an anti-MN antibody, an anti-MUC1 antibody, ananti-MUC16 antibody, an anti-NaPi2b antibody, an anti-Nectin-4 antibody,an anti-PSMA antibody, an anti-SIRPα antibody, an anti-SLC44A4 antibody,an anti-STEAP-1 antibody, an anti-5T4 (or anti-TPBG, trophoblastglycoprotein) antibody, an anti-Tag72 antibody, an anti-TF (oranti-tissue factor) antibody, an anti-TROP2 antibody and an anti-VLAantibody.

Preferably, the antibody is an anti-annexin A1 antibody, an anti-CD115antibody, an anti-CD123 antibody, an anti-CLL-1 antibody, an anti-c-METantibody, an anti-MUC1 antibody, an anti-PSMA antibody, an anti-5T4antibody or an anti-TF antibody. More preferably, the antibody is ananti-PSMA antibody or an anti-5T4 antibody.

The antibody to be used in accordance with the present inventionpreferably is a monoclonal antibody (mAb) and can be a chimeric,humanized or human mAb. More preferably, in accordance with the presentinvention a humanized or human mAb is used, even more preferably ahumanized or human IgG antibody, most preferably a humanized or humanIgG1 mAb. Preferably, said antibody has κ (kappa) light chains, i.e., ahumanized or human IgG1-κ antibody.

In humanized antibodies, the antigen-binding CDRs in the variableregions are derived from antibodies from a non-human species, commonlymouse, rat or rabbit. These non-human CDRs are placed within a humanframework (FR1, FR2, FR3 and FR4) of the variable region, and arecombined with human constant regions. Like human antibodies, thesehumanized antibodies can be numbered according to the Kabat numberingsystem. The present invention particularly relates to an ADC compoundwherein said engineered cysteine is at a position selected from VH 40,VH 41, VH 89, VL 40 or VL 41 in the human framework (i.e., VH 40, VH 41,VL 40 and VL 41 are in the middle of FR2, VH 89 is in FR3) of ahumanized antibody, more particularly at VH 40, VH 41, VL 40 or VL 41,even more particularly at VH 41, VL 40 or VL 41, especially at VH 41.

In accordance with the present invention, these specific residues in theframework regions are both suitable for conjugation of a linker drug anddo not lead to significant reduction of antigen binding properties ofthe antibody after conjugation of the linker drug. Furthermore, thesesites are not only suitable in antibodies, but also in any antigenbinding fragments thereof.

In one particular embodiment, the present invention relates to an ADCcompound as described hereinabove wherein said antibody is ananti-annexin A1 antibody, an anti-CD115 antibody, an anti-CD123antibody, an anti-CLL-1 antibody, an anti-c-MET antibody, an anti-MUC1antibody, an anti-PSMA antibody, an anti-5T4 antibody or an anti-TFantibody and said linker drug comprises a duocarmycin derivative,preferably an ADC compound in accordance with formula (I) or (II).

In a further particular embodiment, the present invention relates to anADC compound as described hereinabove wherein said antibody is ananti-PSMA (monoclonal) antibody or an anti-5T4 (monoclonal) antibody andsaid linker drug comprises a duocarmycin derivative, preferably an ADCcompound in accordance with formula (I) or (II).

In a preferred embodiment, the present invention relates to an ADCcompound as described hereinabove, wherein said linker drug comprises aduocarmycin derivative and is conjugated at position 41 of the heavychain variable region of an anti-PSMA (monoclonal) antibody or ananti-5T4 (monoclonal) antibody, most preferably an ADC compoundaccording to formula (II). Suitable anti-PSMA antibodies are describedin WO98/03873 (e.g., Example 12), WO02/098897 (e.g., FIGS. 1-2),WO2007/002222 (e.g., Table 1) and WO2011/069019 (e.g., FIG. 2). Suitableanti-5T4 antibodies include H8, which is described in WO2006/031653, andthe A1 and A3 antibodies which are described in WO2007/106744, as wellas any antibodies binding to the same epitope as these known antibodies.

In a more preferred embodiment, the heavy chain of the anti-PSMAantibody comprises the amino acid sequence of SEQ ID NO:2 and the lightchain of the anti-PSMA antibody comprises the amino acid sequence of SEQID NO:5. More preferably, the heavy chain of the anti-PSMA antibodycomprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3, andthe light chain of the anti-PSMA antibody comprises the amino acidsequences of SEQ ID NO:5 and SEQ ID NO:6.

In a particularly preferred embodiment, the present invention relates toan ADC compound of formula (II), wherein the antibody is an anti-PSMAantibody, the heavy chain of said anti-PSMA antibody comprising theamino acid sequence of SEQ ID NO:2 and the light chain of said anti-PSMAantibody comprising the amino acid sequence of SEQ ID NO:5. Morepreferably, the heavy chain of said anti-PSMA antibody comprises theamino acid sequences of SEQ ID NO:2 and SEQ ID NO:3, and the light chainof said anti-PSMA antibody comprises the amino acid sequences of SEQ IDNO:5 and SEQ ID NO:6.

In another more preferred embodiment, the heavy chain of the anti-5T4antibody comprises the amino acid sequence of SEQ ID NO:8 and the lightchain of the anti-5T4 antibody comprises the amino acid sequence of SEQID NO:11. More preferably, the heavy chain of the anti-5T4 antibodycomprises the amino acid sequences of SEQ ID NO:8 and SEQ ID NO:9, andthe light chain of the anti-5T4 antibody comprises the amino acidsequences of SEQ ID NO:11 and SEQ ID NO:6.

In a particularly preferred embodiment, the present invention relates toan ADC compound of formula (II), wherein the antibody is an anti-5T4antibody, the heavy chain of said anti-5T4 antibody comprising the aminoacid sequence of SEQ ID NO:8 and the light chain of said anti-5T4antibody comprising the amino acid sequence of SEQ ID NO:11. Morepreferably, the heavy chain of said anti-5T4 antibody comprises theamino acid sequences of SEQ ID NO:8 and SEQ ID NO:9, and the light chainof said anti-5T4 antibody comprises the amino acid sequences of SEQ IDNO:11 and SEQ ID NO:6.

The present invention further relates to an ADC compound as describedhereinabove for use as a medicament.

In one embodiment, the present invention relates to an ADC compound asdescribed hereinabove for use in the treatment of human solid tumoursand haematological malignancies.

In a further embodiment, the present invention relates to an ADCcompound as described hereinabove for use in the treatment of humansolid tumours selected from the group consisting of breast cancer,gastric cancer, colorectal cancer, urothelial cancer (e.g. bladdercancer), ovarian cancer, uterine cancer, lung cancer (especiallynon-small cell lung cancer and small-cell lung cancer), mesothelioma(especially malignant pleural mesothelioma), liver cancer, pancreaticcancer, and prostate cancer.

In a preferred embodiment, the present invention relates to an ADCcompound as described hereinabove, particularly a compound comprising ananti-PSMA (monoclonal) antibody and a duocarmycin derivative linkerdrug, for use in the treatment of prostate cancer.

In another preferred embodiment the present invention relates to an ADCcompound as described hereinabove, particularly a compound comprising ananti-5T4 (monoclonal) antibody and a duocarmycin derivative linker drug,for use in the treatment of human solid tumours selected from the groupconsisting of breast cancer, gastric cancer, colorectal cancer, ovariancancer, lung cancer (especially non-small cell lung cancer (NSCLC) andsmall-cell lung cancer (SCLC)), and malignant pleural mesothelioma.

In yet a further embodiment, the present invention relates to an ADCcompound as described hereinabove for use in the treatment of humanhaematological malignancies, particularly leukaemia, selected from thegroup consisting of acute lymphoblastic and myeloid leukaemia (ALL andAML, respectively).

The present invention further relates to a pharmaceutical compositioncomprising an ADC compound as described hereinabove and one or morepharmaceutically acceptable excipients. Typical pharmaceuticalformulations of therapeutic proteins such as monoclonal antibodies and(monoclonal) antibody-drug conjugates take the form of lyophilized cakes(lyophilized powders), which require (aqueous) dissolution (i.e.,reconstitution) before intravenous infusion, or frozen (aqueous)solutions, which require thawing before use.

Typically, the pharmaceutical composition is provided in the form of alyophilized cake. Suitable pharmaceutically acceptable excipients forinclusion into the pharmaceutical composition (before freeze-drying) inaccordance with the present invention include buffer solutions (e.g.citrate, histidine or succinate containing salts in water),lyoprotectants (e.g. sucrose, trehalose), tonicity modifiers (e.g.sodium chloride), surfactants (e.g. polysorbate), and bulking agents(e.g. mannitol, glycine). Excipients used for freeze-dried proteinformulations are selected for their ability to prevent proteindenaturation during the freeze-drying process as well as during storage.As an example, the sterile, lyophilized powder single-use formulation ofKadcyla™ (Roche) contains—upon reconstitution with Bacteriostatic orSterile Water for Injection (BWFI or SWFI)—20 mg/mL ado-trastuzumabemtansine, 0.02% w/v polysorbate 20, 10 mM sodium succinate, and 6% w/vsucrose with a pH of 5.0.

The present invention also relates to the use of a compound or apharmaceutical composition as described hereinabove for the treatment ofhuman solid tumours and haematological malignancies as describedhereinabove.

The present invention further relates to the use of a sequentially orsimultaneously administered combination of a compound or apharmaceutical composition as described hereinabove with a therapeuticantibody and/or a chemotherapeutic agent, for the treatment of humansolid tumours and haematological malignancies as described hereinabove.

In one embodiment of the present invention, the therapeutic antibody isadecatumumab, alemtuzumab, amatuximab, bevacizumab, cetuximab,denosumab, etaracizumab, farletuzumab, gemtuzumab, labetuzumab,mapatumumab, minretumomab, nimotuzumab, nivolumab, oregovomab,panitumumab, pemtumomab, pertuzumab, ramucirumab, sibrotuzumab,trastuzumab or volociximab and the chemotherapeutic agent is i) analkylating agent, particularly nitrogen mustards, such asmechlorethamine, chlorambucil, cyclophosphamide, ifosfamide andmelphalan, nitrosoureas, such as streptozocin, carmustine and lomustine,alkyl sulfonates, such as busulfan, triazines, such as dacarbazine andtemozolomide, ethylenimines, such as thiotepa and altretamine, orplatinum drugs, such as cisplatin, carboplatin and oxaliplatin, ii) ananti-metabolite, particularly 5-fluorouracil, 6-mercaptopurine,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,hydroxyurea, methotrexate or pemetrexed, iii) an anti-tumour antibiotic,particularly daunorubicin, doxorubicin, epirubicin, idarubicin,actinomycin D, bleomycin, mitomycin-C or mitoxantrone, iv) atopoisomerase inhibitor, particularly topoisomerase I inhibitors, suchas topotecan and irinotecan, or topoisomerase II inhibitors, such asetoposide, teniposide and mitoxantrone, v) a mitotic inhibitor,particularly taxanes, such as paclitaxel, cabazitaxel and docetaxel,epothilones, such as ixabepilone, vinca alkaloids, such as vinblastine,vincristine and vinorelbine, or estramustine, vi) a signalling cascadeinhibitor, particularly mTOR (mammalian target of rapamycin) inhibitors,such as temsirolimus and everolimus, or tyrosine kinase inhibitors, suchas gefitinib, erlotinib, imatinib, pazopanib, ceritinib, crizotinib,lapatinib and afatinib, vii) a corticosteroid, particularly prednisone,methylprednisolone or dexamethasone, viii) a hormonal therapeutic agent,particularly androgen receptor modulating agents, such as bicalutamide,enzalutamide and abiraterone acetate, anti-oestrogens, such astamoxifen, or aromatase inhibiting or steroid modifying agents, such asanastrozole, letrozole, fulvestrant and exemestane, ix) a PARPinhibitor, particularly olaparib, or x) another chemotherapy drug,particularly L-asparaginase or bortezomib. The person skilled in the artwill have no difficulty in selecting suitable combination therapies foruse in the treatment of human solid tumours and haematologicalmalignancies as described hereinabove.

In another embodiment of the present invention, particularly in case ofan anti-PSMA ADC compound comprising a duocarmycin derivative linkerdrug, the therapeutic antibody is bevacizumab, denosumab, pertuzumab ortrastuzumab and the chemotherapeutic agent is a topoisomerase IIinhibitor, particularly mitoxantrone, a mitotic inhibitor, particularlya taxane, more particularly cabazitaxel or docetaxel, a corticosteroid,particularly prednisone, or a hormonal therapeutic agent, particularlyan androgen receptor modulating agent, more particularly enzalutamide orabiraterone acetate.

In yet another embodiment of the present invention, particularly in caseof an anti-5T4 ADC compound comprising a duocarmycin derivative linkerdrug, the therapeutic antibody is bevacizumab, cetuximab, nivolumab, orramucirumab and the chemotherapeutic agent is an alkylating agent,particularly a platinum drug, more particularly cisplatin orcarboplatin, an anti-metabolite, particularly gemcitabine or pemetrexed,a topoisomerease II inhibitor, particularly etoposide, a mitoticinhibitor, particularly a taxane or a vinca alkaloid, more particularlypaclitaxel or docetaxel, or vinblastine or vinorelbine, or a signallingcascade inhibitor, particularly a tyrosine kinase inhibitor, moreparticularly erlotinib, ceritinib, crizotinib or afatinib.

In a further embodiment of the present invention, particularly in caseof an anti-5T4 ADC compound comprising a duocarmycin derivative linkerdrug, the therapeutic antibody is bevacizumab and the chemotherapeuticagent is an alkylating agent, particularly a nitrogen mustard, aplatinum drug or a triazine, more particularly cyclophosphamide,ifosfamide, cisplatin, or temozolomide, an anti-tumour antibiotic,particularly doxorubicin, an anti-metabolite, particularly gemcitabine,a topoisomerease I or II inhibitor, particularly topotecan, irinotecanor etoposide, or a mitotic inhibitor, particularly a taxane or a vincaalkaloid, more particularly paclitaxel or docetaxel, or vincristine orvinorelbine.

In yet a further embodiment of the present invention, particularly incase of an anti-5T4 ADC compound comprising a duocarmycin derivativelinker drug, the therapeutic antibody is amatuximab and thechemotherapeutic agent is an alkylating agent, particularly a platinumdrug, more particularly cisplatin or carboplatin, an anti-metabolite,particularly gemcitabine or pemetrexed, or a mitotic inhibitor,particularly a vinca alkaloid, more particularly vinorelbine.

A therapeutically effective amount of the compounds in accordance withthe present invention lies in the range of about 0.01 to about 15 mg/kgbody weight, particularly in the range of about 0.1 to about 10 mg/kgbody weight, more particularly in the range of about 0.3 to about 10mg/kg body weight. This latter range corresponds roughly to a flat closein the range of 20 to 800 mg of the ADC compound. The compound of thepresent invention may be administered weekly, bi-weekly, three-weekly ormonthly, for example weekly for the first 12 weeks and then every threeweeks until disease progression. Alternative treatment regimens may beused depending upon the severity of the disease, the age of the patient,the compound being administered, and such other factors as would beconsidered by the treating physician.

EXAMPLES

Transient Expression of Engineered Cysteine (Mutant) Antibodies

1a) Preparation of cDNAs

The cDNA sequence for the heavy chain was obtained from the amino acidsequences of a leader sequence (SEQ ID NO:1), the heavy chain variableregion of an anti-PSMA antibody (SEQ ID NO:2, Kabat numbering, having acysteine residue at position 41) and the human IgG1 heavy chain constantregion (SEQ ID NO:3, sequential numbering, Eu numbering starting atalanine 118) by back-translating the combined amino acid sequences intoa cDNA sequence optimized for expression in human cells (Homo sapiens)(SEQ ID NO:4).

Similarly, the cDNA sequence for the light chain was obtained from theamino acid sequences of a secretion signal (SEQ ID NO:1), the lightchain variable region of an anti-PSMA antibody (SEQ ID NO:5, Kabatnumbering), and the human κ Ig light chain constant region (SEQ ID NO:6,sequential numbering) by back-translating the combined amino acidsequences into a cDNA sequence optimized for expression in human cells(Homo sapiens) (SEQ ID NO:7).

Similarly, the cDNA sequence for the heavy chain of the anti-5T4antibody H8-HC41 (SEQ ID NO:10) was obtained from the amino acidsequences of a leader sequence (SEQ ID NO:1), the heavy chain variableregion of the H8 antibody (SEQ ID NO:8, sequential numbering, having acysteine residue at position 41) and the human IgG1 heavy chain constantregion (SEQ ID NO:9, sequential numbering, Eu numbering starting atalanine 118).

The cDNA sequence for the H8 antibody light chain (SEQ ID NO:12) wasobtained from the amino acid sequences of a leader sequence (SEQ IDNO:1), the light chain variable region of the H8 antibody (SEQ ID NO:11,Kabat numbering), and the human κ Ig light chain constant region (SEQ IDNO:6, sequential numbering).

The cDNA sequence for the heavy chain of natalizumab (SEQ ID NO:16) wasobtained from the amino acid sequences of a leader sequence (SEQ IDNO:13), the heavy chain variable region of natalizumab (SEQ ID NO:14,Kabat numbering) and the human IgG4 heavy chain constant region (SEQ IDNO:15, sequential numbering, Eu numbering starting at alanine 118;having a proline residue at position 225 and a cysteine residue atposition 375).

The cDNA sequence for the natalizumab light chain (SEQ ID NO:19) wasobtained from the amino acid sequences of a leader sequence (SEQ IDNO:17), the light chain variable region of natalizumab (SEQ ID NO:18,Kabat numbering), and the human κ Ig light chain constant region (SEQ IDNO:6, sequential numbering).

The heavy chain and light chain cDNA constructs were chemicallysynthesized by and obtained from a commercial source (LifeTechnologies). Cleavage of the leader sequence corresponded to thepredicted cleavage site using the SignalP program(http://www.cbs.dtu.dk/services/SignalP/).

1b) Vector Construction and Cloning Strategy

For expression of the antibody chains the mammalian expression vector0098 was constructed as follows. The CMV:BGHpA expression cassette wasexcised from the pcDNA3.1(−) (Life Technologies) plasmid and re-insertedback into the same original vector (still containing an intact CMV:BGHpAexpression cassette), thereby duplicating the CMV:BGHpA expressioncassette, to allow expression of both HC and LC cDNAs from a singleplasmid vector. Subsequently, an IRES-DHFR fragment was isolated fromthe vector pOptiVEC-TOPO (Life Technologies) and inserted between theCMV promoter and the BGHpA polyadenylation sequence in one of theCMV:BGHpA expression cassettes.

The cDNAs for the heavy chain (HC) and the light chain (LC) were ligatedinto pMA-RQ and pMA-T plasmid vectors (Life Technologies), respectively,using SfiI restriction sites. After transfer to E. coli K12 andexpansion, the LC cDNA was transferred to the mammalian expressionvector 0098 using AscI and HpaI restriction sites. The resulting vectorwas digested with BamHI and NheI restriction enzymes, and ligated withthe HC cDNA fragment, digested with the same restriction enzymes. Thefinal vector, containing both the HC and LC expression cassettes(CMV:HC:BGHpA and CMV:LC-BGHpA, respectively) was transferred to andexpanded in E. coli NEB 5-alpha cells (New England Biolabs).

Large-scale production of the final antibody mutant expression vectorfor transfection was performed using Maxi- or Megaprep kits (Qiagen).

2) Transient Expression in Mammalian Cells

Commercially available Expi293F cells (Life Technologies) weretransfected with the antibody mutant expression vector prepared under 1)above using the ExpiFectamine transfection agent (Life Technologies)according to the manufacturer's instructions as follows: 75×10⁷ cellswere seeded in 300 mL Expi293 Expression medium; 300 μg of the antibodymutant expression vector was combined with 800 μl of ExpiFectaminetransfection agent and added to the cells. One day after transfection,1.5 mL Enhancer 1 and 15 mL Enhancer 2 were added to the culture. Sixdays post transfection, the cell culture supernatant was harvested bycentrifugation at 4,000 g for 15 minutes and filtering the clarifiedharvest over MF 75 filters (Nalgene).

3) Purification of Expressed Proteins

Clarified harvests were first checked on expression level using SDS-PAGEelectrophoresis. As production was deemed adequate, the antibodies werepurified using commercially available protein A resin (MabSelect SuRe,GE Healthcare), using Äkta chromatographic equipment (GE Healthcare). A20 cm bed height column was used with a maximum load of 25 mg/mL packedresin. The process was performed at RT.

After equilibration (PBS pH7.4) and loading the purification stepemployed two wash steps (PBS pH7.4 and NaAc pH5, respectively) and anelution step (25 mM NaAc, pH3) followed by a regeneration, rinse andcleaning step, respectively, before a new cycle was started. During theelution step the target protein was collected in a peak starting andstopping at an absorbance of 0.05-0.1 AU (0.2 cm cell length). Afterpurification the protein was stored at −20° C. to −80° C.

4) Concentration and Buffer Exchange to the ADC Conjugation Buffer

Protein A eluates were, if needed, concentrated to 20-25 mg/mL usingVivaspin centrifugal devices (5 or 30 kDa cut-off, Vivaproducts). Afterobtaining the desired concentrations the concentrated solutions(typically 25 mg/mL) were dialyzed twice using PD10 columns (GEHealthcare) and 4.2 mM L-Histidine+50 mM Trehalose pH6.0 buffer.Alternatively, when protein A eluate concentrations were approximately10 mg/mL, no concentration step was employed and the eluate wasimmediately dialyzed three times using snakeskin dialysis tube (10 kDacut-off, Thermo Scientific) against 4.2 mM L-Histidine+50 mM TrehalosepH6.0 buffer. Any precipitate that appeared was removed by filtrationafter dialysis was completed. Concentrations were measured by UVspectroscopy using either Nanodrop or a cuvette UV spectrophotometer(both Thermo Scientific). Quality analysis showed that the protein had apurity of >95% and contained negligible amounts of dimers or fragmentsas determined by HPSEC. The isoelectric point of the engineered cysteinemutant was identical to the wild-type.

Using the similar/same procedure as described hereinabove for thepreparation and purification of the engineered cysteine (VH 41C, Kabatnumbering) anti-PSMA antibody, the engineered H8-HC41 (VH 41C, Kabatnumbering) and the engineered cysteine natalizumab (CH 225P, 375C, Kabatnumbering) antibodies, also the other antibodies of the examples wereprepared and purified.

General Site-Specific Conjugation Protocol

To a solution of cysteine engineered antibody (5-10 mg/ml in 4.2 mMhistidine, 50 mM trehalose, pH 6) EDTA (25 mM in water, 4% v/v) wasadded. The pH was adjusted to ˜7.4 using TRIS (1 M in water, pH 8) afterwhich TCEP (10 mM in water, 20 equivalents) was added and the resultingmixture was incubated at room temperature for 1-3 hrs. The excess TCEPwas removed by either a PD-10 desalting column or a Vivaspin centrifugalconcentrator (30 kDa cut-off, PES) using 4.2 mM histidine, 50 mMtrehalose, pH 6. The pH of the resulting antibody solution was raised to˜7.4 using TRIS (1 M in water, pH 8) after which dehydroascorbic acid(10 mM in water, 20 equivalents) was added and the resulting mixture wasincubated at room temperature for 1-2 hrs. DMA was added followed by asolution of linker drug (10 mM in DMA). The final concentration of DMAwas 5-10%. The resulting mixture was incubated at room temperature inthe absence of light for 1-16 hrs. In order to remove the excess oflinker drug, activated charcoal was added and the mixture was incubatedat room temperature for 1 hr. The coal was removed using a 0.2 μm PESfilter and the resulting ADC was formulated in 4.2 mM histidine, 50 mMtrehalose, pH 6 using a Vivaspin centrifugal concentrator (30 kDacut-off, PES). Finally, the ADC solution was sterile filtered using a0.22 μm PES filter.

General Conjugation Protocol for Conjugation Via Partially ReducedEndogenous Disulfides (Wt Conjugation)

To a solution of antibody (5-10 mg/ml in 4.2 mM histidine, 50 mMtrehalose, pH 6) EDTA (25 mM in water, 4% v/v) was added. The pH wasadjusted to ˜7.4 using TRIS (1 M in water, pH 8) after which TCEP (10 mMin water, 1-3 equivalents depending on the antibody and the desired DAR)was added and the resulting mixture was incubated at room temperaturefor 1-3 hrs. DMA was added followed by a solution of linker drug (10 mMin DMA). The final concentration of DMA was 5-10%. The resulting mixturewas incubated at room temperature in the absence of light for 1-16 hrs.In order to remove the excess of linker drug, activated charcoal wasadded and the mixture was incubated at room temperature for 1 hr. Thecoal was removed using a 0.2 μm PES filter and the resulting ADC wasformulated in 4.2 mM histidine, 50 mM trehalose, pH 6 using a Vivaspincentrifugal concentrator (30 kDa cut-off, PES). Finally, the ADCsolution was sterile filtered using a 0.22 μm PES filter.

Using the above general procedures, cysteine engineered and wild-typeADCs based on vc-seco-DUBA (SYD980; i.e., compound 18b, n=1 in Example10 on page 209 of WO2011/133039), vc-MMAE and mc-MMAF linker drugs weresynthesized and characterized using analytical Hydrophobic InteractionChromatography (HIC), Size Exclusion Chromatography (SEC), ShieldedHydrophobic Phase Chromatography (SHPC), RP-HPLC and LALendotoxin-testing.

For analytical HIC, 5-10 μL of sample (1 mg/ml) was injected onto aTSKgel Butyl-NPR column (4.6 mm ID×3.5 cm L, Tosoh Bioscience, cat. nr.14947). The elution method consisted of a linear gradient from 100%Buffer A (25 mM sodium phosphate, 1.5 M ammonium sulphate, pH 6.95) to100% of Buffer B (25 mM sodium phosphate, pH 6.95, 20% isopropanol) at0.4 ml/min over 20 minutes. A Waters Acquity H-Class UPLC systemequipped with PDA-detector and Empower software was used. Absorbance wasmeasured at 214 nm and the retention time of ADCs was determined.

As made apparent by analytical HIC, there were differences in theretention times (RTs) for the DAR2 species of the different cysteineengineered ADCs versus the wt conjugates (Tables 1, 2 and 3). Mostinterestingly, conjugating the linker drug at specific sites inside theFab cavity or Fc cavity (as predicted by the molecular modellingalgorithm) gave rise to a (dramatic) decrease in the retention time ascompared to the ADCs that were conjugated via partially reducedendogenous disulfides, leading the present inventors to conclude thatbased on the HIC data, the engineered ADCs in which the linker drug isconjugated to specific sites in the Fab or Fc cavity are lesshydrophobic than the wt conjugates. To further quantify this effect, theterm relative hydrophobicity is introduced, which is defined as:(RT_(DAR2)−RT_(DAR0))/(RT_(DAR2,wt-ADC)−RT_(DAR0,wt-ADC)).In essence, the relative hydrophobicity is a measure that allows afacile comparison between the hydrophobicity of the engineered ADCsversus the wt-conjugated counterparts based on HIC data. The data aresummarized in Tables 1, 2 and 3.

TABLE 1 The relative hydrophobicity of vc-seco-DUBA ADCs on thepreviously specified analytical HIC column: ADC (vc-seco- Cys mutationsHMW Relative DUBA) HC LC DAR (%)² RT_(DAR2) RT_(DAR0) hydrophobicity³ADC-wt wt wt 1.8 7.7 9.7 6.9 1.0 (SYD998)¹ ADC-HC41 S41C wt 1.7 1.4 8.56.8 0.6 (SYD1091) ADC-HC120 T120C wt 1.8 0.9 11.3 6.8 1.6 (SYD1035)⁶ADC-HC152 E152C wt 1.5 1.2 8.5 6.5 0.7 ADC-HC153 P153C wt 1.5 2.4 8.76.5 0.8 ADC-HC236⁶ G236C wt 1.0 1.1 10.4 6.5 1.4 ADC-HC247 P247C wt 1.31.3 9.2 7.3 0.7 ADC-HC339 A339C wt 1.7 0.5 8.6 7.3 0.5 ADC-HC375 S375Cwt 1.8 1.0 7.5 6.6 0.3 ADC-HC376 D376C wt 1.4 3.1 9.8 6.6 1.1 ADC-HC41-S41C, wt 3.3 40.0 12.3 7.3 0.9 120⁷ T120C ADC-HC41- S41C, wt 3.0-4.3⁴1.9 9.3⁵ 7.3 0.4 375 S375C ADC-LC40 wt P40C 1.8 0.5 9.5 6.9 0.9 ADC-LC41wt G41C 1.8 0.6 8.7 6.9 0.6 ADC-LC109⁶ wt T109C 1.0 — 12.4 7.2 1.9ADC-LC154⁶ wt L154C 1.7 6.4 12.4 6.8 2.0 ADC-LC157⁶ wt G157C 1.1 — 12.57.1 1.9 ADC-LC165 wt E165C 1.5 2.3 8.4 6.6 0.6 ADC-LC205⁶ wt V205C 1.81.0 10.6 6.9 1.3 H8-wt¹ wt wt 2.0 4.4 9.9 6.4 1.0 H8-HC40 S40C wt 1.71.2 8.8 6.2 0.7 H8-HC41 P41C wt 1.7 0.4 7.4 6.2 0.3 Natalizumab- S375C⁸wt 1.7 26.0 7.8 6.8 0.4 HC375 ¹Random-non-site specific-attachment ²HMWare high molecular weight species, reflecting the amount of aggregatesformed ³Defined as (RT_(DAR2) − RT_(DAR0))/(RT_(DAR2, wt-ADC) −RT_(DAR0, wt-ADC)), RT is retention time ⁴Based on MS-data ⁵RT, wt DAR4species = 12.2 ⁶Comparator ADCs with linker drug conjugated to acysteine residue pointing outwards ⁷ADC with linker drug conjugated toone cysteine in the Fab cavity and one cysteine residue pointingoutwards; process not yet optimised ⁸Also 225P mutation to preventdimerisation of IgG4

TABLE 2 The relative hydrophobicity of vc-MMAE ADCs on the previouslyspecified analytical HIC column: Relative Cys hydro- ADC (vc- mutationsHMW pho- MMAE) HC LC DAR (%)¹ RT_(DAR2) RT_(DAR0) bicity² ADC-wt wt wt1.7 0.6 9.6 7.2 1.0 ADC-HC41 S41C wt 1.8 0.5 8.1 7.2 0.4 ADC-LC40 wtP40C 1.8 0.6 8.5 7.2 0.5 ADC-LC41 wt G41C 1.9 0.9 8.4 7.3 0.5 H8-HC40S40C wt 1.7 1.4 8.1 6.5 ND³ ¹HMW are high molecular weight species,reflecting the amount of aggregates formed ²Defined as (RT_(DAR2) −RT_(DAR0))/(RT_(DAR2, wt-ADC) − RT_(DAR0, wt-ADC)), RT is retention time³ND is not determined; wild-type H8-vc-MMAE was not prepared

TABLE 3 The relative hydrophobicity of mc-MMAF ADCs on the previouslyspecified analytical HIC column: Relative Cys hydro- ADC (mc- mutationsHMW pho- MMAF) HC LC DAR (%)¹ RT_(DAR2) RT_(DAR0) bicity² ADC-wt wt wt1.8 0.6 8.0 7.2 1.0 ADC-HC41 S41C wt 1.8 0.5 7.4 7.2 0.3 ADC-LC40 wtP40C 1.8 0.5 7.6 7.2 0.5 ADC-LC41 wt G41C 1.8 0.6 7.5 7.3 0.3 H8-wt wtwt 4.2 0.4 7.2 6.2 1.0 H8-HC40 S40C wt 1.4 1.2 6.9 6.5 0.4 ¹HMW are highmolecular weight species, reflecting the amount of aggregates formed²Defined as (RT_(DAR2) − RT_(DAR0))/(RT_(DAR2, wt-ADC) −RT_(DAR0, wt-ADC)), RT is retention time

Cellular Binding

Three anti-PSMA ADCs SYD998 (wt), SYD1091 (HC41) and comparator SYD1035(HC120) had equal binding affinities on PSMA-expressing LNCaP-C4.2 cells(EC₅₀ in the range of 0.1-0.2 μg/ml) similar to the wild-type antibody,and all three ADCs were unable to bind to PSMA-negative DU-145 cells(EC₅₀>10 μg/ml).

Two anti-5T4 ADCs H8-wt and H8-HC40 had equal binding affinities on5T4-expressing MDA-MB-468 cells (EC₅₀ in the range of 0.1-1.2 μg/ml)similar to the wild-type H8 antibody, and both ADCs were unable to bindto 5T4-negative SK-MEL-30 cells (EC₅₀>10 μg/ml).

The antigen binding properties of the ADCs were thus unaffected by theattached duocarmycin derivative linker drug.

In Vitro Cytotoxicity

The potencies of the site-specifically conjugated anti-PSMA ADCs weresimilar to the conventionally linked wild-type ADC (SYD998) onPSMA-expressing LNCaP-C4.2 cells (IC₅₀ in the range of 0.1-0.5 nM, basedon drug equivalents, see Table 4 below). All ADCs were inactive onPSMA-negative DU-145 cells (IC₅₀>70 nM) indicating selective killing oftumour cells through PSMA.

The two non-binding control ADCs were at least 50-times less potent thanthe anti-PSMA ADCs on each of the cell lines evaluated.

TABLE 4 In vitro cytotoxicity of the anti-PSMA-vc-seco- DUBA ADCs inhuman tumour cells expressing PSMA PSMA-positive cell line LNCaP-C4.2ADC (vc- Cys mutations IC₅₀ 95% CI seco-DUBA) HC LC (nM) (nM)¹ %efficacy² ADC-wt wt wt 0.23 0.20-0.27 82 (SYD998) ADC-HC41 S41C wt 0.250.21-0.28 78 (SYD1091) ADC-HC120 T120C wt 0.14 0.13-0.16 82 (SYD1035)ADC-HC152 E152C wt 0.44 0.36-0.55 78 ADC-HC153 P153C wt 0.34 0.28-0.4179 ADC-HC236 G236C wt 0.22 0.19-0.26 76 ADC-HC247 P247C wt 0.100.09-0.12 82 ADC-HC339 A339C wt 0.12 0.11-0.13 83 ADC-HC375 S375C wt0.25 0.22-0.28 81 ADC-HC376 D376C wt 0.20 0.18-0.22 82 ADC-LC40 wt P40C0.30 0.23-0.37 80 ADC-LC41 wt G41C 0.31 0.25-0.38 80 ADC-LC154 wt L154C0.24 0.19-0.29 82 ADC-LC165 wt E165C 0.51 0.40-0.65 79 ADC-LC205 wtV205C 0.17 0.15-0.19 83 Non-binding wt wt 28.86 24.76-36.02 96control-wt Non-binding P41C wt >100 n.a. n.a. control-HC41 Free linker0.02 0.02-0.03 98 drug ¹95% CI is 95% confidence interval ²Percentageefficacy was given at the highest concentration tested (~100 nM) andcalculated by dividing the measured luminescence for each drug or ADCwith the average mean of untreated cells (only growth medium) multipliedby 100.

Conjugation of vc-MMAE to the HC41, LC40 and LC41 positions on anti-PSMAantibodies resulted in cytotoxic potencies in PSMA-positive LNCaP-C4.2cells similar to anti-PSMA-vc-seco-DUBA linked on these cysteine sites(Tables 4 and 5). The anti-PSMA-vc-MMAE ADCs lacked activity onPSMA-negative DU-145 cells (IC₅₀>70 nM).

TABLE 5 In vitro cytotoxicity of the anti-PSMA-vc-MMAE ADCs in humantumour cells expressing PSMA PSMA-positive cell line LNCaP-C4.2 ADC Cysmutations IC₅₀ 95% CI (vc-MMAE) HC LC (nM) (nM)¹ % efficacy² ADC-wt wtwt 0.34 0.31-0.38 89 ADC-HC41 S41C wt 0.39 0.35-0.43 91 ADC-LC40 wt P40C0.31 0.27-0.35 90 ADC-LC41 wt G41C 0.33 0.29-0.37 90 Non-binding wtwt >100 n.a. 90 control-wt Free linker 0.23 0.18-0.28 94 drug ¹95% CI is95% confidence interval ²Percentage efficacy was given at the highestconcentration tested (~100 nM) and calculated by dividing the measuredluminescence for each drug or ADC with the average mean of untreatedcells (only growth medium) multiplied by 100.

The potencies of the engineered anti-5T4 ADCs were equal to theconventionally linked ADC H8-wt on 5T4-expressing MDA-MB-468 cells (IC₅₀between 0.07 and 0.09 nM, Table 6). The anti-5T4 ADCs were inactive on5T4-negative SK-MEL-30 cells (IC₅₀>90 nM).

TABLE 6 In vitro cytotoxicity of the anti-5T4-vc-seco- DUBA ADCs inhuman tumour cells expressing 5T4 vc-seco-DUBA 5T4-positive cell lineMDA-MB-468 ADC (vc- Cys mutations IC₅₀ 95% CI seco-DUBA) HC LC (nM)(nM)¹ % efficacy² H8-wt wt wt 0.09 0.08-0.10 91 H8-HC40 S40C wt 0.070.07-0.08 88 H8-HC41 P41C wt 0.07 0.06-0.08 88 Non-binding P41C wt 40.1834.24-47.16 85 control-HC41 Free linker 0.02 0.01-0.02 94 drug ¹95% CIis 95% confidence interval ²Percentage efficacy was given at the highestconcentration tested (~100 nM) and calculated by dividing the measuredluminescence for each drug or ADC with the average mean of untreatedcells (only growth medium) multiplied by 100.

Together, these data show that the tested cysteine positions forsite-specific conjugation did not have an impact on the tumour cellkilling potency of ADCs comprising two different linker drugs. Moreover,site-specific linkage of linker drugs in the variable region of the Fabpart of different antibodies is generally applicable.

Enzymatic Cleavage by Cathepsin B

The valine-citrulline moiety present in the linker of the ADCs withvc-seco-DUBA (SYD980) and vc-MMAE can be cleaved by cysteine proteases,such as cathepsin B, which results in subsequent intracellular releaseof the (seco-)DUBA or MMAE drug inside the tumour lysosomes orextracellular in the tumour microenvironment. To assess the sensitivitytowards cathepsin B, the ADCs were treated for 2 minutes and 4 hourswith activated cathepsin B (Calbiochem). The cytotoxic activity of thereleased drug from the anti-PSMA ADCs was measured on PSMA-negativeDU-145 cells. The cytotoxic activity of the released drug from theanti-5T4 ADCs was measured on 5T4-negative SK-MEL-30 cells. During thepre-incubation step at 37° C., 1 mg/ml of each ADC was mixed with 5μg/ml cathepsin B (0.04 units/well) in 0.1M Na-acetate pH 5 containing 4mM DTT. As a control, 1 mg/ml of each ADC was directly diluted inculture medium (RPMI 1640, 10% qualified FBS). Serial dilutions weremade from these ADC solutions in culture medium. To measure release ofthe respective free toxins DUBA or MMAE, PSMA-negative DU-145 cells(1,000 cells/well) and 5T4-negative SK-MEL-30 cells (2,000 cells/well)were cultured with the ADCs for 6 days, and the cell viability wasmeasured after 6 days using the CellTiter-Glo™ (CTG) assay kit.

Differences in potency of the released drug on PSMA-negative DU-145cells and 5T4-negative SK-MEL-30 cells reflect the amount of drug thatis cleaved from the ADC, and thereby the accessibility of thevaline-citrulline cleavage site for cathepsin B. As shown in Table 7,the sensitivity for proteolytic cleavage differed amongst the ADCs afterfour hours of exposure to activated cathepsin B (see IC₅₀ values), whilenone of the ADCs were cleaved after a short period of 2 minutes exposurewith cathepsin B (IC₅₀>10 nM, data not shown in Table).

Together these data show that the site of conjugation influences theaccessibility of the linker drug for enzymatic cleavage, and that thevc-seco-DUBA (SYD980) linker drug in the anti-PSMA ADCs ADC-HC41,ADC-HC152, ADC-HC339, ADC-HC375, ADC-LC41, and ADC-LC165 are mostshielded from cleavage by said enzyme. Conjugation of vc-MMAE to theHC41 and LC41 positions of the anti-PSMA antibodies resulted in similarshielding of the valine-citrulline cleavage site (Table 7). A similartrend was also seen for the anti-5T4 antibody H8-HC41 conjugated tovc-seco-DUBA (SYD980) via the same HC41 position.

These data together show that particularly the 41C position is asuitable position for site-specific conjugation of linker-drugs tovarious antibodies.

TABLE 7 Cytotoxicity of the free drug cleaved by cathepsin B 4 hourspre-incubation with cathepsin B Cys mutations IC₅₀ 95% CI ADC HC LC (nM)(nM) % efficacy anti-PSMA antibodies conjugated to vc-seco-DUBA ADC-wtwt wt 0.70 0.62-0.79 97 (SYD998) ADC-HC41 S41C wt ~5.00 n.a. 59(SYD1091) ADC-HC120 T120C wt 0.38 0.34-0.42 96 (SYD1035) ADC-HC152 E152Cwt >10 n.a. 50 ADC-HC153 P153C wt 0.76 0.69-0.84 98 ADC-HC236 G236C wt2.08 1.64-2.65 100  ADC-HC247 P247C wt 2.01 1.69-2.39 99 ADC-HC339 A339Cwt 5.00 3.50-7.15 99 ADC-HC375 S375C wt >10 n.a. 45 ADC-HC376 D376C wt0.60 0.52-0.68 98 ADC-LC40 wt P40C 2.11 1.91-2.34 96 ADC-LC41 wtG41C >10 n.a. n.a. ADC-LC154 wt L154C 0.26 0.22 0.30 98 ADC-LC165 wtE165C >10 n.a. 50 ADC-LC205 wt V205C 0.48 0.41-0.58 97 Non-binding wt wt0.32 0.28-0.35 97 control-wt Non-binding P41C wt 1.56 1.36-1.79 98control- HC41 anti-PSMA antibodies conjugated to vc-MMAE ADC-wt wt wt0.63 0.31-0.38 96 ADC-HC41 S41C wt 2.28 2.11-2.46 97 ADC-LC40 wt P40C0.60 0.55-0.64 96 ADC-LC41 wt G41C 4.28 3.65-5.02 96 Non-binding wt wt0.64 0.57-0.72 97 control-wt anti-5T4 antibodies conjugated tovc-seco-DUBA H8-wt wt wt 0.35* 0.30-0.40 98 H8-HC40 S40C wt 0.980.83-1.15 93 H8-HC41 P41C wt 1.27* 0.98-1.67 98 Non-binding P41C wt 1.861.42-2.45 85 control- HC41 *LNCaP-C4.2 was used as the 5T4-negative cellline.

Tumour Xenograft Animal Model

The in vivo efficacy of three anti-PSMA ADCs was evaluated in the LNCaPC4-2 prostate cancer xenograft model. The LnCaP-C4.2 cell line is ahuman prostate carcinoma epithelial cell line derived from a xenograftthat was serially propagated in mice after castration-induced regressionand relapse of the parental, androgen-dependent LnCaP-FGC xenograft cellline.

Tumours were induced subcutaneously by injecting 1×10⁷ of LnCap C4.2cells in 200 μL of RPMI 1640 containing matrigel (50:50, v:v) into theright flank of male CB17-SCID mice. LnCaP-C4.2 tumour cell implantationwas performed 24 to 72 hours after a whole body irradiation with aγ-source (1.44 Gy, ⁶⁰Co, BioMep, Bretenières, France). Treatments werestarted when the tumours reached a mean volume of 100-200 mm³. Mice wererandomized according to their individual tumour volume into groups andreceived a single i.v. injection of anti-PSMA ADC (2 or 10 mg/kg) orvehicle in the tail vein. Changes in tumour volumes (FIG. 2) and bodyweight (FIG. 3) were monitored. All three ADCs have an average DAR ofapproximately 1.8.

FIG. 2A demonstrates that at 2 mg/kg the comparator engineered cysteineanti-PSMA ADC SYD1035 is less active compared to the native,non-engineered cysteine SYD998. However, the efficacy of SYD1091, anengineered cysteine ADC in accordance with the present invention, issignificantly better than the comparator SYD1035 and is better than thenative, non-engineered SYD998. The difference between the comparatorSYD1035 and SYD1091 is even more pronounced at 10 mg/kg as shown in FIG.2B. Mice bearing LnCap C4.2 tumours develop cachexia as illustrated inFIG. 3. This loss of body weight is often restored after administrationof efficacious treatments and is considered a sensitive efficacybiomarker. Treatment with SYD1091 resulted in much faster restoration ofthe body weights than was seen with the comparator SYD1035 or native,non-engineered SYD998 (FIG. 3).

The in vivo efficacy of two anti-5T4 ADCs, i.e. the nativeH8-vc-seco-DUBA (average DAR 2.0) and the engineered cysteine (VH P41C)ADC H8-41C-vc-seco-DUBA (average DAR 1.7), was evaluated in the PA-1ovarian cancer xenograft model. The PA-1 cell line was established fromcells taken from ascitic fluid collected from a woman with ovariancarcinoma (Zeuthen J. et al. Int. J. Cancer 1980; 25(1): 19-32).

PA-1 tumours were induced subcutaneously by injecting 1×10⁷ cells in 100μL RPMI 1640 medium containing matrigel (50/50, v/v) into the rightflank of female Balb/c nude mice. PA-1 tumour cell injection wasperformed 24 to 72 hours after a whole body irradiation with a γ-source(2 Gy, ⁶⁰Co, BioMep, Bretenières, France). Treatments were started whenthe tumours reached a mean volume of 200-300 mm³. Mice were randomizedaccording to their individual tumour volume into groups and received asingle i.v. injection of anti-5T4 ADC (3 or 10 mg/kg) or vehicle in thetail vein and changes in tumour volumes (FIGS. 4A and 4B) weremonitored. Even though both variants have similar efficacy at the higherclose 10 mg/kg (FIG. 4B), at 3 mg/kg the engineered cysteine anti-5T4ADC H8-41C-vc-seco-DUBA was clearly more active when compared to thenative, non-engineered anti-5T4 ADC, H8-vc-seco-DUBA (FIG. 4A).

Together these findings demonstrate that, in vivo, the site-specificengineered cysteine ADCs according to the present invention showfavourable properties with respect to the efficacy in mouse tumourmodels.

(HAVT20 leader sequence) SEQ ID NO: 1    1 MACPGFLWAL VISTCLEFSM A(anti-PSMA antibody HC S41C) SEQ ID NO: 2    1EVQLVQSGAE VKKPGASVKI SCKTSGYTFT EYTIHWVKQA CGKGLEWIGN   51INPNNGGTTY NQKFEDRATL TVDKSTSTAY MELSSLRSED TAVYYCAAGW  101NFDYWGQGTT VTVSS (human IgG1 antibody HC constant region) SEQ ID NO: 3   1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV   51HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP  101KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS  151HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK  201EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC  251LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW  301QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (anti-PSMA antibody HC S41C cDNA)SEQ ID NO: 4    1ATGGCCTGTC CTGGATTTCT GTGGGCCCTC GTGATCAGCA CCTGTCTGGA ATTCAGCATG   61GCCGAGGTGC AGCTGGTGCA GTCTGGCGCC GAAGTGAAGA AACCAGGCGC CAGCGTGAAG  121ATCAGCTGCA AGACCAGCGG CTACACCTTC ACCGAGTACA CCATCCACTG GGTCAAGCAG  181GCCTGTGGCA AGGGCCTGGA ATGGATCGGC AACATCAACC CCAACAACGG CGGCACCACC  241TACAACCAGA AGTTCGAGGA CCGGGCCACC CTGACCGTGG ACAAGAGCAC AAGCACCGCC  301TACATGGAAC TGAGCAGCCT GCGGAGCGAG GACACCGCCG TGTACTATTG TGCCGCCGGA  361TGGAACTTCG ACTACTGGGG CCAGGGCACC ACCGTGACAG TGTCTAGCGC CAGCACAAAG  421GGCCCCAGCG TGTTCCCTCT GGCCCCTAGC AGCAAGTCTA CCTCTGGCGG AACAGCCGCC  481CTGGGCTGCC TCGTGAAGGA CTACTTTCCC GAGCCCGTGA CCGTGTCCTG GAACTCTGGC  541GCTCTGACAA GCGGCGTGCA CACCTTTCCA GCCGTGCTGC AGAGCAGCGG CCTGTACTCT  601CTGAGCAGCG TCGTGACTGT GCCCAGCAGC AGCCTGGGCA CCCAGACCTA CATCTGCAAC  661GTGAACCACA AGCCCAGCAA CACCAAGGTG GACAAAAAGG TGGAACCCAA GAGCTGCGAC  721AAGACCCACA CCTGTCCCCC TTGTCCTGCC CCTGAACTGC TGGGCGGACC TTCCGTGTTC  781CTGTTCCCCC CAAAGCCCAA GGACACCCTG ATGATCAGCC GGACCCCCGA AGTGACCTGC  841GTGGTGGTGG ATGTGTCCCA CGAGGACCCT GAAGTGAAGT TCAATTGGTA CGTGGACGGC  901GTGGAAGTGC ACAACGCCAA GACCAAGCCC AGAGAGGAAC AGTACAACAG CACCTACCGG  961GTGGTGTCCG TGCTGACAGT GCTGCACCAG GACTGGCTGA ACGGCAAAGA GTACAAGTGC 1021AAGGTGTCCA ACAAGGCCCT GCCTGCCCCC ATCGAGAAAA CCATCAGCAA GGCCAAGGGC 1081CAGCCCCGCG AACCCCAGGT GTACACACTG CCTCCCAGCA GGGACGAGCT GACCAAGAAC 1141CAGGTGTCCC TGACATGCCT CGTGAAAGGC TTCTACCCCT CCGATATCGC CGTGGAATGG 1201GAGAGCAACG GCCAGCCCGA GAACAACTAC AAGACCACCC CCCCTGTGCT GGACAGCGAC 1261GGCTCATTCT TCCTGTACAG CAAGCTGACT GTGGATAAGT CCCGGTGGCA GCAGGGCAAC 1321GTGTTCAGCT GCAGCGTGAT GCACGAGGCC CTGCACAACC ACTACACCCA GAAAAGCCTG 1381TCCCTGAGCC CCGGCAAG (anti-PSMA antibody LC) SEQ ID NO: 5    1DIVMTQSPSS LSASVGDRVT ITCKASQDVG TAVDWYQQKP GKAPKLLIYW   51ASTRHTGVPD RFTGSGSGTD FTLTISSLQP EDFADYFCQQ YNSYPLTFGG  101 GTKLEIK(human IgG antibody LC κ constant region) SEQ ID NO: 6    1RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG   51NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK  101 SFNRGEC(anti-PSMA antibody LC cDNA) SEQ ID NO: 7    1ATGGCTTGTC CTGGATTTCT GTGGGCCCTC GTGATCAGCA CCTGTCTGGA ATTCAGCATG   61GCCGACATCG TGATGACCCA GAGCCCCAGC TCTCTGAGCG CCAGCGTGGG CGACAGAGTG  121ACCATCACAT GCAAGGCCAG CCAGGACGTG GGCACCGCCG TGGATTGGTA TCAGCAGAAG  181CCTGGCAAGG CCCCCAAGCT GCTGATCTAC TGGGCCAGCA CCAGACACAC CGGCGTGCCC  241GATAGATTCA CAGGCAGCGG CTCCGGCACC GACTTCACCC TGACAATCAG CAGCCTGCAG  301CCCGAGGACT TCGCCGACTA CTTCTGCCAG CAGTACAACA GCTACCCCCT GACCTTCGGC  361GGAGGCACCA AGCTGGAAAT CAAGCGGACA GTGGCCGCTC CCAGCGTGTT CATCTTCCCA  421CCTAGCGACG AGCAGCTGAA GTCTGGCACC GCCTCTGTCG TGTGCCTGCT GAACAACTTC  481TACCCCCGCG AGGCCAAGGT GCAGTGGAAG GTGGACAATG CCCTGCAGAG CGGCAACAGC  541CAGGAAAGCG TGACCGAGCA GGACAGCAAG GACTCCACCT ACAGCCTGAG CAGCACCCTG  601ACCCTGAGCA AGGCCGACTA CGAGAAGCAC AAGGTGTACG CCTGCGAAGT GACCCACCAG  661GGCCTGTCTA GCCCCGTGAC CAAGAGCTTC AACCGGGGCG AGTGC (H8 HC P41C)SEQ ID NO: 8    1 QVQLVQSGAE VKKPGASVKV SCKASGYSFT GYYMHWVKQS CGQGLEWIGR  51 INPNNGVTLY NQKFKDRVTM TRDTSISTAY MELSRLRSDD TAVYYCARST  101MITNYVMDYW GQGTLVTVSS (human IgG1 antibody HC constant region)SEQ ID NO: 9    1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV  51 HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP  101KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS  151HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK  201EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC  251LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW  301QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (H8 HC P41C cDNA) SEQ ID NO: 10    1ATGGCCTGTC CTGGATTTCT GTGGGCCCTC GTGATCAGCA CCTGTCTGGA ATTCAGCATG   61GCCCAGGTGC AGCTGGTGCA GTCTGGCGCC GAAGTGAAGA AACCAGGCGC CAGCGTGAAG  121GTGTCCTGCA AGGCCAGCGG CTACAGCTTC ACCGGCTACT ACATGCACTG GGTCAAGCAG  181AGCTGCGGCC AGGGCCTGGA ATGGATCGGC AGAATCAACC CCAACAACGG CGTGACCCTG  241TACAACCAGA AATTCAAGGA CCGCGTGACC ATGACCCGGG ACACCAGCAT CAGCACCGCC  301TACATGGAAC TGAGCCGGCT GAGAAGCGAC GACACCGCCG TGTACTACTG CGCCCGGTCC  361ACCATGATCA CCAACTACGT GATGGACTAC TGGGGCCAGG GCACCCTCGT GACAGTGTCT  421AGCGCCAGCA CAAAGGGCCC CAGCGTGTTC CCTCTGGCCC CTAGCAGCAA GAGCACATCT  481GGCGGAACAG CCGCCCTGGG CTGCCTCGTG AAGGATTACT TCCCCGAGCC CGTGACCGTG  541TCCTGGAATA GCGGAGCCCT GACAAGCGGC GTGCACACCT TTCCAGCCGT GCTGCAGAGC  601AGCGGCCTGT ACTCTCTGAG CAGCGTCGTG ACTGTGCCCA GCAGCAGCCT GGGCACCCAG  661ACCTACATCT GCAACGTGAA CCACAAGCCC AGCAACACCA AGGTGGACAA GAAGGTGGAA  721CCCAAGAGCT GCGACAAGAC CCACACCTGT CCCCCTTGTC CTGCCCCTGA ACTGCTGGGC  781GGACCTTCCG TGTTCCTGTT CCCCCCAAAG CCCAAGGACA CCCTGATGAT CAGCCGGACC  841CCCGAAGTGA CCTGCGTGGT GGTGGATGTG TCCCACGAGG ACCCTGAAGT GAAGTTCAAT  901TGGTACGTGG ACGGCGTGGA AGTGCACAAC GCCAAGACCA AGCCCAGAGA GGAACAGTAC  961AACAGCACCT ACCGGGTGGT GTCCGTGCTG ACAGTGCTGC ACCAGGACTG GCTGAACGGC 1021AAAGAGTACA AGTGCAAGGT GTCCAACAAA GCCCTGCCTG CCCCCATCGA GAAAACCATC 1081AGCAAGGCCA AGGGCCAGCC CCGCGAACCC CAGGTGTACA CACTGCCTCC CAGCCGGGAA 1141GAGATGACCA AGAACCAGGT GTCCCTGACA TGCCTCGTGA AAGGCTTCTA CCCCTCCGAT 1201ATCGCCGTGG AATGGGAGAG CAACGGCCAG CCCGAGAACA ACTACAAGAC CACCCCCCCT 1261GTGCTGGACA GCGACGGCTC ATTCTTCCTG TACAGCAAGC TGACCGTGGA CAAGTCCCGG 1321TGGCAGCAGG GCAACGTGTT CAGCTGCAGC GTGATGCACG AGGCCCTGCA CAACCACTAC 1381ACCCAGAAGT CCCTGAGCCT GAGCCCCGGC AAA (H8 LC) SEQ ID NO: 11    1DIVMTQSPDS LAVSLGERAT INCKASQSVS NDVAWYQQKP GQSPKLLISY   51TSSRYAGVPD RFSGSGSGTD FTLTISSLQA EDVAVYFCQQ DYNSPPTFGG  101 GTKLEIK(H8 LC cDNA) SEQ ID NO: 12    1ATGGCCTGTC CTGGATTTCT GTGGGCCCTC GTGATCAGCA CCTGTCTGGA ATTCAGCATG   61GCCGACATCG TGATGACCCA GAGCCCCGAT AGCCTGGCCG TGTCTCTGGG AGAGAGAGCC  121ACCATCAACT GCAAGGCCAG CCAGAGCGTG TCCAACGACG TGGCCTGGTA TCAGCAGAAG  181CCCGGCCAGA GCCCTAAGCT GCTGATCTCC TACACCAGCA GCAGATATGC CGGCGTGCCC  241GACAGATTTT CCGGCAGCGG CTCTGGCACC GACTTCACCC TGACAATCAG CTCCCTGCAG  301GCCGAGGACG TGGCCGTGTA CTTCTGTCAG CAAGACTACA ACAGCCCCCC CACCTTCGGC  361GGAGGCACCA AGCTGGAAAT CAAGCGGACA GTGGCCGCTC CCAGCGTGTT CATCTTCCCA  421CCTAGCGACG AGCAGCTGAA GTCCGGCACA GCCTCTGTCG TGTGCCTGCT GAACAACTTC  481TACCCCCGCG AGGCCAAGGT GCAGTGGAAG GTGGACAATG CCCTGCAGAG CGGCAACAGC  541CAGGAAAGCG TGACCGAGCA GGACAGCAAG GACTCCACCT ACAGCCTGAG CAGCACCCTG  601ACCCTGAGCA AGGCCGACTA CGAGAAGCAC AAGGTGTACG CCTGCGAAGT GACCCACCAG  661GGACTGAGCA GCCCTGTGAC CAAGAGCTTC AACCGGGGCG AGTGC(germline leader sequence) SEQ ID NO: 13    1 MDWTWRILFL VAAATGAHS(natalizumab HC) SEQ ID NO: 14    1QVQLVQSGAE VKKPGASVKV SCKASGFNIK DTYIHWVRQA PGQRLEWMGR   51IDPANGYTKY DPKFQGRVTI TADTSASTAY MELSSLRSED TAVYYCAREG  101YYGNYGVYAM DYWGQGTLVT VSS (natalizumab HC S225P, S375C) SEQ ID NO: 15   1 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV   51HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES  101KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED  151PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK  201CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK  251GFYPCDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG  301NVFSCSVMHE ALHNHYTQKS LSLSLGK (natalizumab HC S225P, S375C cDNA)SEQ ID NO: 16    1ATGGACTGGA CCTGGCGCAT CCTGTTTCTG GTGGCCGCTG CTACCGGCGC TCACTCCCAG   61GTGCAGCTGG TGCAGTCTGG CGCCGAAGTG AAGAAACCTG GCGCCTCCGT GAAGGTGTCC  121TGCAAGGCCT CCGGCTTCAA CATCAAGGAC ACCTACATCC ACTGGGTCCG ACAGGCCCCT  181GGACAGCGGC TGGAATGGAT GGGCAGAATC GACCCCGCCA ACGGCTACAC TAAGTACGAC  241CCCAAGTTCC AGGGCAGAGT GACCATCACC GCCGACACCT CCGCCTCCAC AGCCTACATG  301GAACTGTCCT CCCTGCGGAG CGAGGACACC GCCGTGTACT ACTGCGCCAG AGAGGGCTAC  361TACGGCAACT ACGGCGTGTA CGCCATGGAC TACTGGGGCC AGGGCACCCT GGTCACCGTG  421TCCTCCGCTT CCACCAAGGG CCCCTCCGTG TTCCCTCTGG CCCCTTGCTC CCGGTCCACC  481TCCGAGTCTA CCGCCGCTCT GGGCTGCCTG GTCAAGGACT ACTTCCCCGA GCCCGTGACC  541GTGTCCTGGA ACTCTGGCGC CCTGACCTCT GGCGTGCACA CCTTCCCTGC TGTGCTGCAG  601TCCTCCGGCC TGTACTCCCT GTCCTCCGTC GTGACCGTGC CCTCCAGCTC CCTGGGCACC  661AAGACCTACA CCTGTAACGT GGACCACAAG CCCTCCAACA CCAAGGTGGA CAAGCGGGTG  721GAATCTAAGT ACGGCCCTCC CTGCCCCCCC TGCCCTGCCC CTGAATTTCT GGGCGGACCT  781TCCGTGTTCC TGTTCCCCCC AAAGCCCAAG GACACCCTGA TGATCTCCCG GACCCCCGAA  841GTGACCTGCG TGGTGGTGGA CGTGTCCCAG GAAGATCCCG AGGTCCAGTT CAATTGGTAC  901GTGGACGGCG TGGAAGTGCA CAACGCCAAG ACCAAGCCCA GAGAGGAACA GTTCAACTCC  961ACCTACCGGG TGGTGTCCGT GCTGACCGTG CTGCACCAGG ACTGGCTGAA CGGCAAAGAG 1021TACAAGTGCA AGGTGTCCAA CAAGGGCCTG CCCAGCTCCA TCGAAAAGAC CATCTCCAAG 1081GCCAAGGGAC AGCCTCGCGA GCCCCAGGTG TACACCCTGC CTCCAAGCCA GGAAGAGATG 1141ACCAAGAACC AGGTGTCCCT GACCTGTCTG GTCAAGGGCT TCTACCCCTG CGATATCGCC 1201GTGGAATGGG AGTCCAACGG CCAGCCCGAG AACAACTACA AGACCACCCC CCCTGTGCTG 1261GACTCCGACG GCTCCTTCTT CCTGTACTCT CGGCTGACCG TGGACAAGTC CCGGTGGCAG 1321GAAGGCAACG TCTTCTCCTG CTCCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG 1381AAGTCCCTGT CCCTGAGCCT GGGCAAG (germline leader sequence) SEQ ID NO: 17   1 MDMRVPAQLL GLLLLWLRGA RC (natalizumab LC) SEQ ID NO: 18    1DIQMTQSPSS LSASVGDRVT ITCKTSQDIN KYMAWYQQTP GKAPRLLIHY   51TSALQPGIPS RFSGSGSGRD YTFTISSLQP EDIATYYCLQ YDNLWTFGQG  101 TKVEIK(natalizumab LC cDNA) SEQ ID NO: 19    1ATGGACATGA GAGTGCCCGC CCAGCTGCTG GGACTGCTGC TGCTGTGGCT GAGAGGCGCC   61AGATGCGACA TCCAGATGAC CCAGTCCCCC TCCAGCCTGT CCGCCTCCGT GGGCGACAGA  121GTGACCATCA CATGCAAGAC CTCCCAGGAC ATCAACAAGT ACATGGCCTG GTATCAGCAG  181ACCCCCGGCA AGGCCCCTCG GCTGCTGATC CACTACACCT CCGCTCTGCA GCCTGGCATC  241CCCTCCAGAT TCTCCGGCTC CGGCTCTGGC CGGGACTATA CCTTCACCAT CTCCAGTCTG  301CAGCCCGAGG ATATCGCCAC CTACTACTGC CTGCAGTACG ACAACCTGTG GACCTTCGGC  361CAGGGCACCA AGGTGGAAAT CAAGCGGACC GTGGCCGCTC CCTCCGTGTT CATCTTCCCA  421CCCTCCGACG AGCAGCTGAA GTCCGGCACC GCCTCCGTCG TGTGCCTGCT GAACAACTTC  481TACCCCCGCG AGGCCAAGGT GCAGTGGAAG GTGGACAACG CCCTGCAGTC CGGCAACTCC  541CAGGAATCCG TCACCGAGCA GGACTCCAAG GACAGCACCT ACTCCCTGTC TCCACCCTG  601ACCCTGTCCA AGGCCGACTA CGAGAAGCAC AAGGTGTACG CCTGCGAAGT GACCCACCAG  661GGCCTGTCCA GCCCCGTGAC CAAGTCCTTC AACCGGGGCG AGTGC

The invention claimed is:
 1. A method of treating human solid tumors orhaematological malignancies in a patient in need thereof, comprisingadministering to said patient an effective amount of an antibody-drugconjugate compound (ADC); wherein said ADC comprises an antibody orantigen binding fragment thereof, having an engineered cysteine at heavychain position 41 (according to Kabat numbering) and a linker drugconjugated to said antibody or antigen binding fragment through saidengineered cysteine wherein said antibody binds to an antigen targetthat is expressed on the cell surface of said tumor or malignancy. 2.The method according to claim 1, wherein said patient has a solid tumorselected from the group consisting of breast cancer, gastric cancer,colorectal cancer, urothelial cancer, ovarian cancer, uterine cancer,lung cancer, mesothelioma, liver cancer, pancreatic cancer, and prostatecancer.
 3. The method according to claim 1, wherein said patient has ahaematological malignancy, wherein the haematological malignancy isleukaemia.
 4. The method according to claim 1, wherein the antibody orantigen binding fragment of said ADC further comprises an engineeredcysteine at position 375 of the heavy chain (according to Eu numbering)and linker drug is conjugated through said engineered cysteine atposition
 375. 5. The method according to claim 1, wherein said linkerdrug comprises a cytotoxic drug selected from the group consisting ofanthracyclines, duocarmycins, pyrrolobenzodiazepine (PBD) dimers,calicheamicins, maytansinoids, and auristatins.
 6. The method accordingto claim 5, wherein said cytotoxic drug is monomethyl auristatin E(MMAE) or emtansine (DM1).
 7. The method according to claim 5, whereinsaid cytotoxic drug is a duocarmycin derivative.
 8. The method accordingto claim 7, wherein said ADC has the formula (I)

wherein said Antibody is the antibody or antigen binding fragmentthereof that contains the engineered cysteine at heavy chain position41, n is 0, 1, 2, or 3, m represents an average DAR of from 1 to 6, R¹is selected from the group consisting of

y is 1-16, and R² is selected from the group consisting of


9. The method according to claim 8, wherein n is 0 or 1, m represents anaverage DAR of from 1.5 to 2, R¹ is

y is 1-4, and R² is selected from the group consisting of


10. The method according to claim 9, wherein said ADC has the formula(II)


11. The method according to claim 1, wherein said patient has a solidtumor selected from the group consisting of breast cancer, gastriccancer, colorectal cancer, ovarian cancer, non-small cell lung cancer(NSCLC), small-cell lung cancer (SCLC), and malignant pleuralmesothelioma; and wherein said antibody of said ADC is an anti-5T4monoclonal antibody.
 12. The method according to claim 11, wherein saidanti-5T4 monoclonal antibody has a heavy chain that comprises the aminoacid sequence of SEQ ID NO:8 and a light chain that comprises the aminoacid sequence of SEQ ID NO:11.
 13. The method according to claim 11,wherein said ADC has the formula (I)

wherein said Antibody is the anti-5T4 monoclonal antibody that containsthe engineered cysteine at heavy chain position 41, n is 0, 1, 2, or 3,m represents an average DAR of from 1 to 6, R¹ is selected from thegroup consisting of

y is 1-16, and R² is selected from the group consisting of


14. The method according to claim 13, wherein said ADC has the formula(II)


15. The method according to claim 13, which further comprisesadministering to said patient an effective amount of one or more of atherapeutic antibody or a chemotherapeutic agent, or a combinationthereof.
 16. The method according to claim 1, wherein said patient has asolid tumor, wherein the solid tumor is prostate cancer; and whereinsaid antibody of said ADC is an anti-PSMA monoclonal antibody.
 17. Themethod according to claim 16, wherein said anti-PSMA monoclonal antibodyhas a heavy chain that comprises the amino acid sequence of SEQ ID NO:2and a light chain that comprises the amino acid sequence of SEQ ID NO:5.18. The method according to claim 16, wherein said ADC has the formula(I)

wherein said Antibody is the anti-PSMA monoclonal antibody that containsthe engineered cysteine at heavy chain position 41, n is 0, 1, 2, or 3,m represents an average DAR of from 1 to 6, R¹ is selected from thegroup consisting of

y is 1-16, and R² is selected from the group consisting of


19. The method according to claim 18, wherein said ADC has the formula(II)


20. The method according to claim 18, which further comprisesadministering to said patient an effective amount of one or more of atherapeutic antibody or a chemotherapeutic agent, or a combinationthereof.